Type 1 allergies have worldwide importance. Up to 20% of the population in industrialised countries suffer from complaints such as allergic rhinitis, conjunctivitis or bronchial asthma.
These allergies are caused by sources of various origin, such as trees and grasses (pollen), fungi (spores), mites (excrement), cats or dogs. The allergen sources are released directly into the air (pollen, spores) or can reach the air bonded to diesel soot particles (pollen) or house dust (mite excrement, skin particles, hair). Since the allergy-triggering substances are located in the air, the term aeroallergens is also used.
The type 1 allergy-triggering substances are proteins, glycoproteins or polypeptides. After uptake via mucous membranes, these allergens react with the IgE molecules bound to the surface of mast cells in sensitised persons. If these IgE molecules are crosslinked with one another by an allergen, this results in the secretion of mediators (for example histamine, prostaglandins) and cytokines by the effector cell and thus in the corresponding allergic symptoms.
Up to 40% of type 1 allergy sufferers exhibit specific IgE reactivity with pollen extracts of true grasses (Burney et al., 1997, J. Allergy Clin. Immunol. 99:314-322; D'Amato et al., 1998, Allergy 53: 567-578; Freidhoff et al., 1986, J. Allergy Clin Immunology, 78, 1190-2002). The family of the true grasses (Poaceae) encompasses more than 10000 species, many more than 20 of which are hitherto known as triggers of allergic symptoms (Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107; Esch, 2008, Allergens and Allergen Immunotherapy, Clinical Allergy and Immunology Series, 107-126).
Most of the allergy-triggering true grasses belong to the Pooideae sub-family. Besides the grass species occurring as wild forms, such as, for example, Holcus lanatus (velvet grass), Phalaris aquatica (canary grass), Anthoxanthum odoratum (sweet vernal grass), Dactylis glomerata (orchard grass), Festuca pratensis (meadow fescue), Poa pratensis (Kentucky blue grass) or Lolium perenne (rye grass), cultivated cereals, such as Triticum aestivum (wheat), Secale cereale (rye) and Hordeum vulgare (barley), are also known members of this sub-family.
One of the Pooideae species which has been investigated best with respect to its allergens is Timothy grass (Phleum pratense), which is widespread worldwide as a wild plant and also plays a commercial role as a pasture plant and hardy feed grass.
Depending on the relative frequency in a population with which the individual allergen molecules react with the IgE antibodies of allergy sufferers, a distinction is made between major and minor allergens.
Six allergens of Timothy grass can be regarded as major allergens: Phl p 1 (Petersen et al., 1993, J. Allergy Clin. Immunol. 92: 789-796), Phl p 5 (Matthiesen and Löwenstein, 1991, Clin. Exp. Allergy 21: 297-307; Petersen et al., 1992, Int. Arch. Allergy Immunol. 98: 105-109), Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108, 49-54), Phl p 2/3 (Dolecek et al., 1993, FEBS 335 (3): 299-304), Phl p 4 (Haavik et al., 1985, Int. Arch. Allergy Appl. Immunol. 78: 260-268; Valenta et al., 1992, Int. Arch. Allergy Immunol. 97: 287-294; Nandy et al., Biochem. Biophys. Res. Commun., 2005, 337(2): 563-70) and Phl p 13 (Suck et al., 2000, Clin. Exp. Allergy 30: 1395-1402).
The first description of Phl p 6 came as early as 1978. A protein fraction purified from Timothy grass pollen, which was called “Ag19”, contained an allergen with a size of about 15 kDa, which was later classified in the official allergen nomenclature and was continued as Phl p 6 (Løwenstein, 1978, Allergy 33: 30-41; WHO/IUIS Allergen Nomenclature Subcommittee, www.allergen.org). Phl p 6 is classified as a major allergen since Phl p 6-reactive IgE antibodies can be detected in about 70% of grass pollen allergy sufferers. (Rossi et al., 2001, Allergy, 56: 1180-85; Vrtala et al., 1999, J. Immunol. 15; 163:5489-9).
Physicochemical investigations of the allergen from grass pollen extract detected two protein variants which differ in their primary sequence (Blume et al., 2004, Proteomics 4: 1366-71). These isoforms are attributed to two cDNA sequences identified in expression libraries of Timothy grass pollen and carry the WHO/IUIS names Phl p 6.0101 (GenBank: Z27082.1; UniProt: P43215; see FIGS. 15 and 16 or SEQ ID NO:3 and SEQ ID NO: 4, with propeptide see FIG. 19 or SEQ ID NO:9; Petersen et al., 1995, Int. Arch. Allergy Immunol. 108: 55-59) and Phl p 6.0102 (GenBank: Y16955; UniProt: 065868; see FIGS. 3 and 4 or SEQ ID NO:1 and SEQ ID NO: 2, with propeptide see FIG. 20 or SEQ ID NO:10; Vrtala et al., 1999, J. Immunol. 15; 163:5489-9). Apart from a signal peptide, the proteins each consist of 110 amino acids and differ at only two positions (Val 14→Ile and Arg→95 His, starting from ripe Phl p 6.0101), which cause a difference in the molecular weight of 5 Da (11790 Da of Phl p 6.0101 compared with 11785 Da of Phl p 6.0102; FIG. 1).
The pollen of other true grass species of the Poaceae family and in particular the Pooideae sub-family may contain major allergens which are homologous with the allergens of Timothy grass. Such allergens which occur across species are summarised as an allergen group. The high structural homology of such related allergens, which is ultimately based on a similar amino acid sequence, causes correspondingly high cross-reactivity of the molecules with IgE antibodies (Lorenz et al., 2009, Int. Arch. Immunol. 148:1-17). It is also known that atopic persons who react allergically to major allergens of Timothy grass may have been primarily sensitised by one of the other related species of the true grasses. Finally, this cross-reactivity may mean that sensitization by one grass species is sufficient to trigger an allergic reaction by other related grasses.
A group 6 allergen which is cross-reactive with Phl p 6 has already been detected at the protein level in pollen of Kentucky blue grass (Poa pratensis) (Vrtala et al., 1999, J. Immunol. 15; 163:5489-9; Niederberger et al., 1998, J. Allergy Clin. Immunol. 101 (2): 258-264).
Besides the cross-reactivity of the group 6 allergens with one another, cross-reactivity with major allergens from group 5 is also known. The polypeptide chain of Phl p 6 exhibits great similarity with an N-terminal region of Phl p 5, which has a size of about 26-28 kDa (FIG. 1, FIG. 2). It is thought that the allergens can be attributed to a common original gene (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108: 55-59). Both proteins form α-helical secondary structures, but no β-folded sheet structures. X-ray structural analysis has shown that the four α-helices of Phl p 6 fold to form a characteristic helix bundle (RCSB Protein Data Bank entry: 1NLX; Fedorov et al., 2003; FIG. 1), a structure which has also been detected in fragments of Phl p 5 (Rajashankar et al., 2002, Acta Cryst. D58:1175-1181; Maglio et al., 2002, Protein Engineering 15: 635-642; Wald et al., 2007, Clin. Exp. Allergy 37:441-450). The similarity between the allergens has the effect that some of the Phl p 5-reactive IgE antibodies also bind to Phl p 6 (Petersen et al., 1995, Int. Arch. Allergy Immunol. 108: 49-54; Andersson & Lidholm, 2003, Int. Arch. Allergy Immunol. 130:87-107).
Specific immunotherapy (SIT) or hyposensitisation is regarded as an effective approach to the therapeutic treatment of allergies (Fiebig 1995 Allergo J. 4 (6):336-339, Bousquet et al., 1998, J. Allergy Clin. Immunol. 102 (4): 558-562; Cox et al., 2007, J. Allergy Clin. Immunol. 120:S25-85; James & Durham, 2008, Clin. Exp. Allergy 38: 1074-1088).
The classical therapy form of injection therapy (SLIT), in which natural allergen extracts are injected subcutaneously into the patient in increasing doses, has been used successfully for about 100 years. In this therapy, the immune system of the allergy sufferer is repeatedly confronted with allergens, causing reprogramming of the immune system to be achieved together with tolerance of the allergens. After uptake of the antigens from the allergen preparations by antigen-presenting cells, peptides are presented to the antigens on the cell surface. Some particular peptides which contain so-called T-cell epitopes are recognised by antigen-specific T-cells. This binding results, inter alia, in the development of various types of T-cells having a regulatory function. In the course of SIT, the regulatory T-cell response results in tolerance of the allergen, the downregulation of TH2 cytokines, the restoration of the TH1/TH2 equilibrium, the suppression of allergen-specific IgE, the induction of IgG4, IgG1 and IgA antibodies, the suppression of effector cells (mast cells, basophils and eosinophils) and the renewal of inflamed tissue (Akdis et al., 2007, J. Allergy Clin. Immunol. 119 (4):780-789; Larche et al., 2008, Nature Reviews 6:761-771). The T-cell epitopes are thus of crucial importance for the therapeutic action of allergen preparations in the case of hyposensitisation.
Owing to the cross-reactivity of the major allergens of the true grasses which is present at IgE and also at T-cell level, successful therapy with an allergen extract of a single representative grass species is usually sufficient (Mailing et al., 1993, EAACI Position Paper: Immunotherapy, Allergy 48: 9-35; Cox et al., 2007, J Allergy Clin Immunol 120: 25-85).
Besides subcutaneous immunotherapy, a sublingual therapy form, in which the allergens or allergen derivatives are taken up via the oral mucous membrane, is undergoing clinical trials and use as an alternative to injection therapy (James & Durham, 2008, Clin. Exp. Allergy 38: 1074-1088).
A further possibility is treatment with expressible DNA which encodes for the relevant allergens (immunotherapeutic vaccination). Experimental evidence of the allergen-specific influencing of the immune response has been furnished in rodents by injection of allergen-encoding DNA (Hsu et al. 1996, Nature Medicine 2 (5):540-544, Weiss et al., 2006, Int. Arch. Allergy Immunol. 139: 332-345).
In all these therapy forms, there is a fundamental risk of allergic reactions or even anaphylactic shock (Kleine-Tebbe, 2006, Allergologie, 4:135-156). In order to minimise these risks, innovative preparations in the form of allergoids are employed. These are chemically modified allergen extracts which have significantly reduced IgE reactivity, but identical T-cell reactivity compared with the untreated extract (Fiebig 1995 Allergo J. 4 (6):336-339, Kahlert et al., 1999, Int. Arch. Allergy Immunol, 120: 146-157).
Therapy optimisation is possible with allergens prepared by recombinant methods. Defined cocktails of high-purity allergens prepared by recombinant methods, which are optionally matched to the individual sensitisation patterns of the patients, could replace extracts from natural allergen sources, since, apart from the various allergens, the latter contain a relatively large number of immunogenic, but non-allergenic accompanying proteins. Initial clinical studies with recombinant allergens have already been carried out with success (Jutel et al., 2005, J. Allergy Clin. Immunol., 116: 608-613; Valenta & Niederberger, 2007, J. Allergy Clin. Immunol. 119: 826-830).
Realistic prospects which may result in safe hyposensitisation with recombinant expression products are offered specifically by mutated recombinant allergens in which IgE epitopes are modified without impairing the T-cell epitopes which are essential for the therapy (Schramm et al. 1999, J. Immunol. 162:2406-2414). These hypoallergenic proteins could be employed in relatively high doses during SIT without increasing the probability of undesired IgE-promoted side effects.
In the past, such “hypoallergenic” variants with reduced IgE binding have been published for many aeroallergens (inter alia pollen and house dust mite allergens) and food allergens. On the basis of the DNA of unmodified allergens, it has been possible to prepare and express a recombinant DNA, inter alia by fragmentation, oligomerisation, deletions, point mutations or recombination of individual sections of an allergen (DNA shuffling) (Ferreira et al., 2006, Inflamm. & Allergy—Drug Targets 5: 5-14; Bhalla & Singh, 2008, Trends in Biotechnology 26:153-161; Westritschnig et al., 2007, J. Immunol. 179: 7624-7634).
Regarding the group 6 allergens of the grasses, only a single mutation strategy has been published to date, in which the first ninety nucleotides, encoding for amino acids 1-30 of ripe Phl p 6, have been deleted. The molecule was expressed as histidine fusion molecule, purified and investigated with respect to its immunological properties. The N-terminal deletion resulted in reduced IgE binding and reduced ability to be stimulated by basophilic granulocytes (Vrtala et al., 2007, J. Immunol. 179: 1730-1739). In a later paper, the same molecule was connected to a recombinant variant of Phl p 2 to give a hybrid molecule (Linhart et al., 2008, Biol. Chem. 389: 925-933). A mutation strategy based on point mutations, as described for other allergens, has not yet been published hitherto for group 6 grass pollen allergens.
The object on which the present invention is based consisted in the provision of novel variants of group 6 allergens of the Poaceae at the protein and DNA level which are distinguished by reduced IgE reactivity at the same time as substantial retention of the T-cell reactivity and are therefore suitable for curative and preventive specific immunotherapy and immunotherapeutic DNA vaccination.